Reactivity‐Based One‐Pot Synthesis of Oligomannoses: Defining Antigens Recognized by 2G12, a Broadly Neutralizing Anti‐HIV‐1 Antibody

Lee, Hing-Ken; Scanlan, Christopher N.; Huang, Chien Jung; Chang, Aileen Y.; Calarese, D.A.; Dwek, Raymond A.; Rudd, Pauline M.; Burton, Dennis R.; Wilson, Ian A.; Wong, Chi‐Huey

doi:10.1002/anie.200353105

Cited by 124 publications

(46 citation statements)

References 27 publications

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“…The identification of its epitope as a cluster of Manα1→2Man termini within the intrinsic mannose patch of gp120 (17) has encouraged the development of vaccines based around biological (19,27,(31)(32)(33) and synthetic Manα1→2Man clusters (34)(35)(36)(37)(38). A major limitation of any vaccine based on 2G12 recognition is the absence of its cognate epitope on the epidemiologically important clade C. The specific Asn residues required for 2G12 neutralization are absent in clade C isolates, explaining their resistance to 2G12.…”

Section: Discussionmentioning

confidence: 99%

Envelope glycans of immunodeficiency virions are almost entirely oligomannose antigens

Doores

Bonomelli

Harvey

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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317

374

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The envelope spike of HIV is one of the most highly N-glycosylated structures found in nature. However, despite extensive research revealing essential functional roles in infection and immune evasion, the chemical structures of the glycans on the native viral envelope glycoprotein gp120-as opposed to recombinantly generated gp120 -have not been described. Here, we report on the identity of the N-linked glycans from primary isolates of HIV-1 (clades A, B, and C) and from the simian immunodeficiency virus. MS analysis reveals a remarkably simple and highly conserved virus-specific glycan profile almost entirely devoid of medial Golgi-mediated processing. In stark contrast to recombinant gp120, which shows extensive exposure to cellular glycosylation enzymes (>70% complex type glycans), the native envelope shows barely detectable processing beyond the biosynthetic intermediate Man 5 GlcNAc 2 (<2% complex type glycans). This oligomannose (Man 5-9 GlcNAc 2 ) profile is conserved across primary isolates and geographically divergent clades but is not reflected in the current generation of gp120 antigens used for vaccine trials. In the context of vaccine design, we also note that Manα1→2Man-terminating glycans (Man 6-9 GlcNAc 2 ) of the type recognized by the broadly neutralizing anti-HIV antibody 2G12 are 3-fold more abundant on the native envelope than on the recombinant monomer and are also found on isolates not neutralized by 2G12. The Manα1→2-Man residues of gp120 therefore provide a vaccine target that is physically larger and antigenically more conserved than the 2G12 epitope itself. This study revises and extends our understanding of the glycan shield of HIV with implications for AIDS vaccine design.HIV | 2G12 | gp120 | glycosylation | vaccine

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Section: Discussionmentioning

confidence: 99%

Envelope glycans of immunodeficiency virions are almost entirely oligomannose antigens

Doores

Bonomelli

Harvey

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

317

374

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“…Such studies suggest that targeting the conserved oligomannose clusters on gp120 is a potential vaccine strategy. Toward this goal, the fine specificity of 2G12 has been characterized structurally and biochemically with chemically defined oligomannoses and/or related synthetic oligosaccharide clusters by a number of groups (7,15,16,24,28,37,41,53,69). Crystal structures of Fab 2G12 alone and in complex with various oligomannosides, including Man 9 GlcNAc 2 and synthetic glycans, revealed the unconventional architecture of 2G12 in which two Fab fragments interlock via heavy chain variable region (V H ) domain swapping, creating a novel V H -V H interface in addition to the two conventional V H -V L (where L indicates the light chain) binding sites (15,16).…”

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confidence: 99%

A Glycoconjugate Antigen Based on the Recognition Motif of a Broadly Neutralizing Human Immunodeficiency Virus Antibody, 2G12, Is Immunogenic but Elicits Antibodies Unable To Bind to the Self Glycans of gp120

Astronomo¹,

Lee

Scanlan

et al. 2008

J Virol

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108

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The glycan shield of human immunodeficiency virus type 1 (HIV-1) gp120 contributes to viral evasion from humoral immune responses. However, the shield is recognized by the HIV-1 broadly neutralizing antibody (Ab), 2G12, at a relatively conserved cluster of oligomannose glycans. The discovery of 2G12 raises the possibility that a carbohydrate immunogen may be developed that could elicit 2G12-like neutralizing Abs and contribute to an AIDS vaccine. We have previously dissected the fine specificity of 2G12 and reported that the synthetic tetramannoside (Man 4 ) that corresponds to the D1 arm of Man 9 GlcNAc 2 inhibits 2G12 binding to gp120 as efficiently as Man 9 GlcNAc 2 itself, indicating the potential use of Man 4 as a building block for creating immunogens. Here, we describe the development of neoglycoconjugates displaying variable copy numbers of Man 4 on bovine serum albumin (BSA) molecules by conjugation to Lys residues. The increased valency enhances the apparent affinity of 2G12 for Man 4 up to a limit which is achieved at ϳ10 copies per BSA molecule, beyond which no further enhancement is observed. Immunization of rabbits with BSA-(Man 4 ) 14 elicits significant serum Ab titers to Man 4 . However, these Abs are unable to bind gp120. Further analysis reveals that the elicited Abs bind a variety of unbranched and, to a lesser extent, branched Man 9 derivatives but not natural N-linked oligomannose containing the chitobiose core. These results suggest that Abs can be readily elicited against the D1 arm; however, potential differences in the presentation of Man 4 on neoglycoconjugates, compared to glycoproteins, poses challenges for eliciting anti-mannose Abs capable of crossreacting with gp120 and HIV-1.

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“…To mimic the 2G12 epitope, glycoantigens have been constructed by several laboratories through chemical synthesis of mannose oligosaccharides and chemoenzymatic conjugation to different scaffolds (8,9,27,29,41,56). However, to our knowledge, these approaches have yet to elicit antibodies that cross-react with gp120 or neutralize the virus.…”

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confidence: 99%

An Engineered Saccharomyces cerevisiae Strain Binds the Broadly Neutralizing Human Immunodeficiency Virus Type 1 Antibody 2G12 and Elicits Mannose-Specific gp120-Binding Antibodies

Luallen

Lin

et al. 2008

J Virol

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The glycan shield of the human immunodeficiency virus type 1 (HIV-1) envelope (Env) protein serves as a barrier to antibody-mediated neutralization and plays a critical role in transmission and infection. One of the few broadly neutralizing HIV-1 antibodies, 2G12, binds to a carbohydrate epitope consisting of an array of high-mannose glycans exposed on the surface of the gp120 subunit of the Env protein. To produce proteins with exclusively high-mannose carbohydrates, we generated a mutant strain of Saccharomyces cerevisiae by deleting three genes in the N-glycosylation pathway, Och1, Mnn1, and Mnn4. Glycan profiling revealed that N-glycans produced by this mutant were almost exclusively Man 8 GlcNAc 2 , and four endogenous glycoproteins that were efficiently recognized by the 2G12 antibody were identified. These yeast proteins, like HIV-1 gp120, contain a large number and high density of N-linked glycans, with glycosidase digestion abrogating 2G12 cross-reactivity. Immunization of rabbits with whole ⌬och1 ⌬mnn1 ⌬mnn4 yeast cells produced sera that recognized a broad range of HIV-1 and simian immunodeficiency virus (SIV) Env glycoproteins, despite no HIV/SIV-related proteins being used in the immunization procedure. Analyses of one of these sera on a glycan array showed strong binding to glycans with terminal Man␣1,2Man residues, and binding to gp120 was abrogated by glycosidase removal of high-mannose glycans and terminal Man␣1,2Man residues, similar to 2G12. Since S. cerevisiae is genetically pliable and can be grown easily and inexpensively, it will be possible to produce new immunogens that recapitulate the 2G12 epitope and may make the glycan shield of HIV Env a practical target for vaccine development.The development of a human immunodeficiency virus (HIV) vaccine able to induce neutralizing antibodies against a broad spectrum of primary isolates is complicated by the large diversity of HIV type 1 (HIV-1) strains, the continual mutation of the envelope (Env) glycoprotein in the face of immune selective pressure, and the presence of numerous N-linked glycans that mask polypeptide epitopes (7). Indeed, genetic deletion of N-linked carbohydrate sites can greatly increase the sensitivity of HIV-1 to antibody-mediated neutralization (3,12,25,26,34,35). One of the few broadly neutralizing monoclonal antibodies (MAbs) isolated from HIV-1-infected patients, 2G12, circumvents these obstacles by binding to relatively conserved high-mannose-type oligosaccharides exposed on the glycan shield of the gp120 subunit of Env (47, 49, 54). The 2G12 epitope consists of an array of at least three such glycans presented as a dense cluster of terminal mannose sugars (49,54). Crystal structures of the 2G12 Fab in complex with carbohydrates reveal a specificity toward Man␣1,2Man disaccharides, alone or terminally exposed on the D1 and D3 arms of Man 9 GlcNAc 2 (Man9) and Man 8 GlcNAc 2 (Man8) structures, without recognizing other mannose disaccharides, including Man␣1,3Man and Man␣1,6Man (8,9). The relatively conserved nature of t...

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Reactivity‐Based One‐Pot Synthesis of Oligomannoses: Defining Antigens Recognized by 2G12, a Broadly Neutralizing Anti‐HIV‐1 Antibody

Cited by 124 publications

References 27 publications

Envelope glycans of immunodeficiency virions are almost entirely oligomannose antigens

Envelope glycans of immunodeficiency virions are almost entirely oligomannose antigens

A Glycoconjugate Antigen Based on the Recognition Motif of a Broadly Neutralizing Human Immunodeficiency Virus Antibody, 2G12, Is Immunogenic but Elicits Antibodies Unable To Bind to the Self Glycans of gp120

An Engineered Saccharomyces cerevisiae Strain Binds the Broadly Neutralizing Human Immunodeficiency Virus Type 1 Antibody 2G12 and Elicits Mannose-Specific gp120-Binding Antibodies

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